Venous thrombosis/thromboembolism affects over 1 million Americans per year. Thrombus burden (size) predicts long-term outcome. Efforts to reduce venous thrombus burden with the development of new drugs could have a considerable impact on the US population. The defining characteristic of venous red thrombi is their high red blood cell (RBC) content, which was traditionally thought to result from passive trapping of RBCs in the static fibrin clot. However, our new preliminary data indicate that RBCs are retained in clots via an active mechanism that requires the fibrin cross-linking enzyme, factor XIII (FXIII) The goal of this application is to determine the molecular mechanisms by which FXIII, fibrin(ogen), FXIII-mediated fibrin cross-linking, and RBCs contribute to venous thrombosis. Our overall hypotheses are that FXIII activity is required to retain RBCs in venous thrombi, and that blocking or reducing FXIII activity will reduce venous thrombus size. This hypothesis will be addressed in three Specific Aims: 1) Map the fibrinogen residues that mediate FXIII binding and determine the impact of FXIII activation and activity on fibrin network structure and stability, 2) Determine the contributions of blood components (FXIII, RBCs, and fibrinogen) to clot size in a whole blood milieu, and 3) Determine the effect of FXIII inhibitors on the formation and stability of venous thrombi. We will employ biochemical, genetic and pharmacologic tools in vitro and in vivo to define the FXIII-fibrinogen axis and determine the role of FXIII activity and RBC retention in venous thrombosis. Fibrin(ogen)-FXIII interactions will be examined using solution phase binding assays, microscopy, innovative fibrin analytical methods, and thromboelastography. Clot formation will be examined in reconstituted whole blood models using plasmas from healthy individuals and patients with a history of venous thrombosis. Thrombus formation and stability will be examined using venous thrombosis and thrombolysis models, immunohistochemistry, ultrasound imaging, and intravital microscopy. These studies will elucidate the biological role of FXIII activity in venous thrombosis, and define novel roles for fibrin(ogen) and RBCs in venous thrombus formation and stability. The study is highly innovative because it challenges the current paradigm that RBCs are passively trapped in static, fibrin- rich venous thrombi. The proposed research is clinically significant because it may reveal new strategies to reduce venous thrombosis in the US population.